The development of alcohol use disorders follows a transition from social use motivated by hedonic and anxiolytic effects to dependence motivated by increasing withdrawal symptoms and an evolving desire to drink during abstinence. In this latter stage of alcohol dependence, abstinence from drinking is often accompanied by negative emotional symptoms, such as increased anxiety and depression, and the alleviation of these negative emotional states is hypothesized to be a major driving force for continued alcohol consumption. This shift from positive to negative reinforcement mechanisms likely results from enduring changes in CNS function induced by excessive alcohol consumption. Although several signaling systems have been implicated in this process there is still an incomplete understanding of the neural mechanisms underlying alcohol dependence. We have gathered evidence that EtOH consumption increases levels of the endogenous cannabinoid (eCB) 2- arachidonoyl glycerol (2-AG) in rodent brain, while long-term intermittent EtOH exposure down-regulates eCB signaling in brain regions relevant to emotional processing. Dependence-associated anxiety-like behavior and excessive EtOH consumption are reduced by generalized enhancement of eCB tone, though similar manipulations do not produce these effects in non-dependent animals. Based on these findings, we hypothesize that eCB clearance inhibitors have therapeutic value for treating alcohol dependence and alcoholism. This hypothesis will be tested through three Specific Aims. Aim 1 will characterize the ability of highly selective eCB clearance inhibitors to alleviate anxiety-like behavior in EtOH dependent mice throughout a period of protracted withdrawal. Importantly, these experiments will characterize the relative influence of two primary eCB molecules, 2-AG and anandamide (AEA), by selectively inhibiting the distinct hydrolytic mechanisms that clear these lipids from the brain. The experiments in Aim 2 will employ biochemical and neurochemical approaches to characterize the mechanisms contributing to dependence-associated dysregulation of brain eCB signaling. Additional work in this Aim will evaluate the influence of eCB dysregulation on other neurotransmitter systems involved in withdrawal-associated anxiety-like behavior and excessive EtOH consumption (including glutamate, serotonin and norepinephrine). The experiments in Aim 3 will characterize the efficacy of selective eCB clearance inhibitors for reducing high levels of EtOH consumption associated with dependence and protracted withdrawal. These experiments will also characterize the influence of eCB signaling on binge-like ethanol intake in non-dependent mice. Completion of the proposed work is likely to highlight a previously unrecognized mechanism in the etiology of alcohol dependence and may identify novel therapeutic targets for alcoholism. PUBLIC HEALTH RELEVANCE: In alcohol dependent individuals, abstinence from drinking is accompanied by negative emotional symptoms such as anxiety that can be relieved by renewed drinking. This form of "self-medication" is believed to be a driving force for continued excessive alcohol consumption. Preliminary evidence indicates that dysfunctional endocannabinoid signaling contributes to withdrawal-related anxiety and excessive alcohol consumption, and the overall goal of this project is to investigate the therapeutic benefit provided by selective endocannabinoid clearance inhibitors for the treatment of alcohol dependence.